No abstract

Effective immunity requires the innate immune system to distinguish foreign (non-self) nucleic acids from cellular (self) nucleic acids. Cellular double-stranded RNAs (dsRNAs) are edited by the RNA editing enzyme ADAR1 to prevent their dsRNA structure pattern being recognized as viral dsRNA by cytoplasmic dsRNA sensors including MDA5, PKR and ZBP1. A loss of ADAR1-mediated RNA editing of cellular dsRNA activates MDA5. However, additional RNA editing-independent functions of ADAR1 have been proposed, but a specific mechanism has not been delineated. We now demonstrate that the loss of ADAR1-mediated RNA editing specifically activates MDA5, while loss of the cytoplasmic ADAR1p150 isoform or its dsRNA binding activity enabled PKR activation. Deleting both MDA5 and PKR resulted in complete rescue of the embryonic lethality of Adar1p150-/- mice to adulthood, contrasting with the limited or no rescue by removing MDA5, PKR or ZBP1 alone, demonstrating that this is a species conserved function of ADAR1p150. Our findings demonstrate that MDA5 and PKR are the primary in vivo effectors of fatal autoinflammation following the loss of ADAR1p150.

ADAR1 -mediated A-to-I RNA editing is a self-/non-self-discrimination mechanism for cellular double-stranded RNAs. ADAR mutations are one cause of Aicardi-Gouti eres Syndrome, an inherited paediatric encephalopathy, classed as a "Type I interferonopathy." The most common ADAR1 mutation is a proline 193 alanine (p.P193A) mutation, mapping to the ADAR1p150 isoform-specific Za domain. Here, we report the development of an independent murine P195A knock-in mouse, homologous to human P193A. The Adar1 P195A/P195A mice are largely normal and the mutation is well tolerated. When the P195A mutation is compounded with an Adar1 null allele (Adar1 P195A/À ), approximately half the animals are runted with a shortened lifespan while the remaining Adar1 P195A/À animals are normal, contrasting with previous reports. The phenotype of the Adar1 P195A/À animals is both associated with the parental genotype and partly non-genetic/ environmental. Complementation with an editing-deficient ADAR1 (Adar1 P195A/E861A ), or the loss of MDA5, rescues phenotypes in the Adar1 P195A/À mice.

The RNA editing enzyme Adenosine deaminase acting on RNA 1 (ADAR1) is an essential regulator of innate immune activation by both cellular and viral dsRNA. Adenosine-to-Inosine (A-to-I) editing by ADAR1 modifies the sequence and structure of endogenous dsRNA and masks it from the cytoplasmic dsRNA sensor melanoma differentiation-associated protein 5 (MDA5), preventing innate immune activation. Loss of function mutations in ADAR are associated with rare autoinflammatory disorders including Aicardi-Goutieres Syndrome (AGS), defined by a constitutive systemic upregulation of type I interferon (IFN). The murine Adar gene encodes two protein isoforms with distinct functions: ADAR1p110 is constitutively expressed and localizes to the nucleus, whereas ADAR1p150 is primarily cytoplasmic and is inducible by IFN. Recent studies have demonstrated the critical requirement for ADAR1p150 to suppress innate immune activation by self dsRNAs, however, detailed in vivo characterization of the role of ADAR1p150 during development and in adult mice is lacking. We developed a new ADAR1p150-specific knockout mouse mutant based on a single nucleotide deletion that resulted in the loss of the ADARp150 protein without affecting ADAR1p110 expression. The Adar1p150-/- died embryonically at E11.5-E12.5 due to cell death in the fetal liver accompanied by an activated IFN response. Somatic loss of ADAR1p150 in adults was lethal and caused rapid hematopoietic failure, demonstrating an ongoing requirement for ADAR1p150 in vivo. The generation and characterization of this mouse model demonstrates the essential role of ADAR1p150 in vivo and provides a new tool for dissecting the functional differences between ADAR1 isoforms and their physiological contributions.